The invention described herein may be manufactured and used by or for the Government of the United States for all governmental purposes without the payment of any royalty.
The present invention relates to biological production of ortho-aminophenols from nitroaromatic compounds
Nitroaromatic compounds are used in the production of dyes, plastics, high explosives, pharmaceuticals and pesticides. Nitrobenzene (NB) alone is discharged to the environment at a rate of tens of millions of pounds annually. In addition, nitrated polycyclic aromatic hydrocarbons are formed during a variety of combustion processes and are common environmental contaminants. Considerable effort has been expended to clean environmentally contaminated sites at or near explosives production facilities. Reduction of the nitro group is a common first step in the biotransformation of nitroaromatic compounds, whether leading to mineralization of the compound or to the accumulation of dead-end products, many of which are cytotoxic and/or mutagenic.
Several biodegradation studies have shown that Pseudomonas pseudoalcaligenes strain JS45 and a variety of other isolates grow on nitrobenzene as the sole source of carbon and nitrogen. The reaction proceeds via two initial intermediates, hydroxylaminobenzene and ortho-aminophenol. The enzymes involved in catalyzing the initial steps are a nitroreductase and a hydroxylaminobenzene mutase. On first inspection the reaction seems similar to the nonenzymatic Bamberger rearrangement. The mechanism of the reactions and the stereochemistry of the products are distinctively different, however. The nitroreductase from strain JS45 has been purified and characterized as a flavoprotein requiring NADPH as an electron donor. Two genes expressing hydroxylaminobenzene mutase activity, have been cloned from strain JS45 and expressed in E. coli (J. K. Davis et al, Appl. Environ. Microbiol., Vol. 66, No. 7, 2965-2971, 2000), and one mutase enzyme, HabB (Z. He et al, Eur. J. Biochem., Vol. 267, 1110-1116, 2000), has been partially purified. JS45 was deposited in the American Type Culture Collection Patent Deposit in January, 2002, Patent Deposit Designation PTA-3972. Other reports have demonstrated that bacteria, such as Clostridium acetobutylicum, Raistonia eutrophus JMP134, Pseudomonas putida, Pseudomonas putida HS12, strain LW1 of the Comamonadaceae family, and Pseudomonas putida 2NP8, synthesize nitroreductases and hydroxylaminoarene mutases that transform a wide range of nitroaromatic compounds to the corresponding aminophenols. The metabolic degradation processes in the above strains are comparable to that described for P. pseudoalcaligenes strain JS45. It should be noted that these studies have been directed to biodegradation, i.e, the breakdown of organic compounds into more cell biomass and less complex compounds, and ultimately to water, and either carbon dioxide or methane. The above-described intermediates have been proposed or noted as intermediates in the biodegradation process(es), and have not been seen as end-products per se.
Orthoaminophenols are important intermediates in the synthesis of common azo dyes and phenoxazinones. They are a key feedstock for the synthesis of polybenzoxazole polymers. The substituents carried by the ortho-aminophenol confer to the benzoxazole products properties that are useful in electronic, opto-electronic, pharmaceutical, medical, military and biosynthetic applications. Commercially useful substituted ortho-aminophenols are difficult to chemically synthesize.
The common route for commercial synthesis of aminophenols occurs in two steps, nitration of phenol followed by reduction of the nitro-group with a metal to make the amine. The influence of the hydroxyl moiety varies with each substrate. For example, for phenol the substitution is directed preferentially to the ortho position, but for naphthalene the para position is more readily attacked. In either case, yields are very low for mononitration of phenols and conditions needed are extreme.
Accordingly, it is an object of the present invention to provide a process for the biological production of aminophenols.
Other objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
In accordance with the present invention there is provided a process for the production of ortho-aminophenols from nitroarenes using a biocatalyst consisting of pure enzymes, partially purified enzymes, cell lysate, intact cells, or a metal reaction linked with a subsequent enzymatic reaction. The biocatalyst is an enzyme system that makes use of a nitroreductase enzyme that initially reduces the nitroarene to the hydroxylaminoarene and a mutase enzyme that converts the hydroxylaminoarene to an ortho-aminophenol. As noted previously, a variety of bacteria produce these enzymes and can be used as the source(s) for the enzymes. The biocatalyst can also consist of a coupled, two-step metal and enzyme reaction in which the metal, such as zinc, catalyzes the transformation of the nitroarene to the hydroxylaminoarene and the mutase then catalyzes the transformation of hydroxylaminoarene to the corresponding ortho-aminophenol.
The reaction scheme can be represented as follows: 
wherein R is selected from the group consisting of xe2x80x94H, xe2x80x94OH, xe2x80x94COOH, xe2x80x94CnH2n+1, xe2x80x94C6H5, xe2x80x94X, xe2x80x94CX3, xe2x80x94CHO, xe2x80x94OCnH2n+1, and xe2x80x94Oxe2x80x94C6H5, wherein n ranges from 1 to 6, X is F, Cl, Br or I, A represents, but is not limited to, a nitroreductase or a metal such as zinc, and B represents the mutase enzyme.